Methods of assaying receptor activity and constructs useful in such methods

ABSTRACT

Described are methods of detecting G-protein coupled receptor (GPCR) activity in vivo and in vitro; methods of assaying GPCR activity; and methods of screening for GPCR ligands, G protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process. Constructs useful in such methods are described.

This application is a continuation of application Ser. No. 09/772,644filed Jan. 30, 2001, now U.S. Pat. No. 6,770,449 entitled Methods ofAssaying Receptor Activity and Constructs Useful in Such Methods; whichis a continuation of application Ser. No. 09/469,554 filed Dec. 22,1999, now U.S. Pat. No. 6,528,271 entitled Inhibition of β-arrestinMediated Effects Prolongs and Potentiates Opioid Receptor-MediatedAnalgesia; which is a continuation in part of Ser. No. 09/233,530, filedJan. 20, 1999, entitled Methods of Assaying Receptor Activity andConstructs Useful in Such Methods, now U.S. Pat. No. 6,110,693, issuedAug. 29, 2000; which is a continuation of Ser. No. 08/869,568, filedJun. 5, 1997, entitled Methods of Assaying Receptor Activity andConstructs Useful in Such Methods, now U.S. Pat. No. 5,891,646, issuedApr. 6, 1999, the contents of which are hereby incorporated by referencein their entireties.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under NationalInstitutes of Health Grant No. HL 03422-02, HL 16037, and NS 19576. TheGovernment has certain rights to this invention.

FIELD OF THE INVENTION

This invention relates to methods of detecting G-protein coupledreceptor (GPCR)activity in vivo and in vitro, and provides methods ofassaying GPCR activity, and methods of screening for GPCR ligands, Gprotein-coupled receptor kinase (GRK) activity, and compounds thatinteract with components of the GPCR regulatory process. This inventionalso provides constructs useful in such methods.

BACKGROUND OF THE INVENTION

The actions of many extracellular signals are mediated by theinteraction of G-protein coupled receptors (GPCRs) and guaninenucleotide-binding regulatory proteins (G proteins). G protein-mediatedsignaling systems have been identified in many divergent organisms, suchas mammals and yeast. GPCRs respond to, among other extracellularsignals, neurotransmitters, hormones, odorants and light. GPCRs aresimilar and possess a number of highly conserved amino acids; the GPCRsare thought to represent a large ‘superfamily’ of proteins. IndividualGPCR types activate a particular signal transduction pathway; at leastten different signal transduction pathways are known to be activated viaGPCRs. For example, the beta 2-adrenergic receptor (βAR) is a prototypemammalian GPCR. In response to agonist binding, βAR receptors activate aG protein (G_(s)) which in turn stimulates adenylate cyclase and cyclicadenosine monophosphate production in the cell.

It has been postulated that members of the GPCR superfamily desensitizevia a common mechanism involving G protein-coupled receptor kinase (GRK)phosphorylation followed by arrestin binding. Gurevich et al., J. Biol.Chem; 270:720 (1995); Ferguson et al., Can. J. Physiol. Pharmacol.74:1095 (1996). However, the localization and the source of the pool ofarrestin molecules targeted to receptors in response to agonistactivation was unknown. Moreover, except for a limited number ofreceptors, a common role for β-arrestin in GPCR desensitization had notbeen established. The role of β-arrestins in GPCR signal transductionwas postulated primarily due to the biochemical observations.

Many available therapeutic drugs in use today target GPCRs, as theymediate vital physiological responses, including vasodilation, heartrate, bronchodilation, endocrine secretion, and gut peristalsis. See,eg., Lefkowitz et al., Ann. Rev. Biochem. 52:159 (1983). GPCRs includethe adrenergic receptors (alpha and beta); ligands to beta ARs are usedin the treatment of anaphylaxis, shock, hypertension, hypotension,asthma and other conditions. Additionally, spontaneous activation ofGPCRs occurs, where a GPCR cellular response is generated in the absenceof a ligand. Increased spontaneous activity can be decreased byantagonists of the GPCR (a process known as inverse agonism); suchmethods are therapeutically important where diseases cause an increasein spontaneous GPCR activity.

Efforts such as the Human Genome Project are identifying new GPCRs(‘orphan’ receptors) whose physiological roles and ligands are unknown.It is estimated that several thousand GPCRs exist in the human genome.With only about 10% of the human genome sequenced, 250 GPCRs have beenidentified; fewer than 150 have been associated with ligands.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a conjugate of a β-arrestinprotein and a detectable molecule. The detectable molecule may be anoptically detectable molecule, such as Green Fluorescent Protein.

A further aspect of the present invention is a nucleic acid constructcomprising an expression cassette. The construct includes, in the 5′ to3′ direction, a promoter and a nucleic acid segment operativelyassociated with the promoter, and the nucleic acid segment encodes aβ-arrestin protein and detectable molecule. The detectable molecule maybe an optically detectable molecule such as Green Fluorescent Protein.

A further aspect of the present invention is a host cell containing anucleic acid molecule which includes, a promoter operable in the hostcell and a nucleic acid sequence encoding a β-arrestin protein and adetectable molecule. The detectable molecule may be an opticallydetectable molecule such as Green Fluorescent Protein. The cell may be amammalian, bacterial, yeast, fungal, plant or animal cell, and may bedeposited on a substrate.

A further aspect of the present invention is a method of assessing Gprotein coupled receptor (GPCR) pathway activity under test conditions,by providing a test cell that expresses a GPCR and that contains aconjugate of a β-arrestin protein and a visually detectable molecule;exposing the test cell to a known GPCR agonist under test conditions;and then detecting translocation of the detectable molecule from thecytosol of the test cell to the membrane edge of the test cell.Translocation of the detectable molecule in the test cell indicatesactivation of the GPCR pathway. Exemplary test conditions include thepresence in the test cell of a test kinase and/or a test G-protein, orexposure of the test cell to a test ligand, or co-expression in the testcell of a second receptor.

A further aspect of the present invention is a method for screening aβ-arrestin protein (or fragment of a β-arrestin protein) for the abilityto bind to a phosphorylated GPCR. A cell is provided that expresses aGPCR and contains a conjugate of a test β-arrestin protein and avisually detectable molecule. The cell is exposed to a known GPCRagonist and then translocation of the detectable molecule from the cellcytosol to the cell edge is detected. Translocation of the detectablemolecule indicates that the β-arrestin molecule can bind tophosphorylated GPCR in the test cell.

A further aspect of the present invention is a method to screen a testcompound for G protein coupled receptor (GPCR) agonist activity. A testcell is provided that expresses a GPCR and contains a conjugate of aβ-arrestin protein and a visually detectable molecule. The cell isexposed to a test compound, and translocation of the detectable moleculefrom the cell cytosol to the membrane edge is detected. Movement of thedetectable molecule to the membrane edge after exposure of the cell tothe test compound indicates GPCR agonist activity of the test compound.The test cell may express a known GPCR or a variety of known GPCRs, orexpress an unknown GPCR or a variety of unknown GPCRS. The GPCR may be,for example, an odorant GPCR or a β-adrenergic GPCR. The test cell maybe a mammalian, bacterial, yeast, fungal, plant or animal cell.

A further aspect of the present invention is a method of screening asample solution for the presence of an agonist to a G protein coupledreceptor (GPCR). A test cell is provided that expresses a GPCR andcontains a conjugate of a β-arrestin protein and a visually detectablemolecule. The test cell is exposed to a sample solution, andtranslocation of the detectable molecule from the cell cytosol to themembrane edge is assessed. Movement of the detectable molecule to themembrane edge after exposure to the sample solution indicates the samplesolution contains an agonist for a GPCR expressed in the cell.

A further aspect of the present invention is a method of screening atest compound for G protein coupled receptor (GPCR) antagonist activity.A cell is provided that expresses a GPCR and contains a conjugate of aβ-arrestin protein and a visually detectable molecule. The cell isexposed to a test compound and to a GPCR agonist, and translocation ofthe detectable molecule from the cell cytosol to the membrane edge isdetected. When exposure to the agonist occurs at the same time as orsubsequent to exposure to the test compound, movement of the detectablemolecule from the cytosol to the membrane edge after exposure to thetest compound indicates that the test compound is not a GPCR antagonist.

A further aspect of the present invention is a method of screening atest compound for G protein coupled receptor (GPCR) antagonist activity.A test cell is provided that expresses a GPCR and contains a conjugateof a β-arrestin protein and a visually detectable molecule. The cell isexposed to a GPCR agonist so that translocation of the detectablemolecule from the cytosol of the cell to the membrane edge of the celloccurs, and the cell is then exposed to a test compound. Where exposureto the agonist occurs prior to exposure to the test compound, movementof the detectable molecule from the membrane edge of the cell to thecytosol after exposure of the cell to the test compound indicates thatthe test compound has GPCR antagonist activity.

A further aspect of the present invention is a method of screening acell for the presence of a G protein coupled receptor (GPCR). A testcell is provided that contains a conjugate of a β-arrestin protein and avisually detectable molecule. The test cell is exposed to a solutioncontaining a GPCR agonist. Any translocation of the detectable moleculefrom the cytosol to the membrane edge is detected; movement of thedetectable molecule from the cytosol to the membrane edge after exposureof the test cell to GPCR agonist indicates that the test cell contains aGPCR.

A further aspect of the present invention is a method of screening aplurality of cells for those cells which contain a G protein coupledreceptor (GPCR). A plurality of test cells containing a conjugate of aβ-arrestin protein and a visually detectable molecule are provided, andthe test cells are exposed to a known GPCR agonist. Cells in which thedetectable molecule is translocated from the cytosol to the membraneedge are identified or detected. Movement of the detectable molecule tothe membrane edge after exposure to a GPCR agonist indicates that thecell contains a GPCR responsive to that GPCR agonist. The plurality oftest cells may be contained in a tissue, an organ, or an intact animal.

A further aspect of the present invention is a substrate havingdeposited thereon a plurality of cells that express a GPCR and thatcontain a conjugate of β-arrestin protein and a detectable molecule.Such substrates may be made of glass, plastic, ceramic, semiconductor,silica, fiber optic, diamond, biocompatible monomer, or biocompatiblepolymer materials.

A further aspect of the present invention is an apparatus fordetermining GPCR activity in a test cell. The apparatus includes meansfor measuring indicia of the intracellular distribution of a detectablemolecule, and a computer program product that includes a computerreadable storage medium having computer-readable program code meansembodied in the medium. The computer-readable program code meansincludes computer-readable program code means for determining whetherthe indicia of the distribution of the detectable molecule in a testcell indicates concentration of the detectable molecule at the cellmembrane, based on comparison to the measured indicia of theintracellular distribution of a detectable molecule in a control cell.The indicia of the intracellular distribution of the detectable moleculemay be optical indicia, and the measuring means may be means formeasuring fluorescent intensity. The molecule to be detected may be onethat is fluorescently detectable, and the step of measuring the indiciaof the intracellular distribution of the detectable molecule may includemeasurement of fluorescence signals from test and control cells.

A further aspect of the present invention is an apparatus fordetermining GPCR activity in a test cell. The apparatus includes meansfor measuring indicia of the intracellular distribution of a detectablemolecule in at least one test cell at multiple time points, and acomputer program product. The computer program product includes acomputer readable storage medium having computer-readable program codemeans embodied in said medium. The computer-readable program code meansincludes computer-readable program code means for determining whetherthe indicia of the distribution of the detectable molecule in the testcell at multiple time points indicates translocation of the detectablemolecule to the cell membrane.

A further aspect of the present invention is an apparatus fordetermining GPCR activity in a test cell, which includes means formeasuring indicia of the intracellular distribution of a detectablemolecule in at least one test cell, and a computer program product. Thecomputer program product includes a computer readable storage mediumhaving computer-readable program code means embodied therein andincluding computer-readable program code means for determining whetherthe indicia of the distribution of the detectable molecule in the testcell indicates concentration of the detectable molecule at the cellmembrane, based on comparison to pre-established criteria.

Pain perception (nociception) is mediated by a cascade of events fromthe point of the stimulus to integrative circuits in the brain.Nociception involves signals that are mediated by several classes ofreceptors and signal transduction mechanisms such as GPCRs for substanceP, opioid peptides, etc. and ion channels such as NMDA receptors.Antinociception has been known for more than 1000 years to be induced bythe alkaloid compound, morphine, which functions as an agonist at the μopioid receptor. The activity of agonists for signaling through GPCRs isusually limited by cellular mechanisms that dampen the signal of theagonist, a process referred to as desensitization. These mechanismsinclude phosphorylation of agonist-activated receptors by specificreceptor kinases called GRKs followed by the interaction of thephosphorylated GPCR with any of the members of the arrestin family ofproteins. Morphine-mediated antinociception is known to wane with time,however the contribution of the desensitization is controversial and forall practical purposes is unknown. With the βarrestin knockout micedisclosed herein, it is shown that interfering with (eliminating) one ofthe key protein components of the desensitization mechanism greatlyenhances the potency and efficacy of the antinociceptive properties ofmorphine.

Accordingly, an additional aspect of the present invention is a knockoutmouse useful for testing the efficacy of potential analgesic agents, thecells of said mouse containing at least one inactive endogenousβarrestin gene (preferably the βarrestin-2 gene), the mouse exhibiting aphenotype of decreased sensitivity to pain after administration of a μopioid receptor agonist such as morphine as compared to thecorresponding wild type mouse. The mouse may be heterozygous orhomozygous for the inactive endogenous βarrestin gene. The mouse isuseful for evaluating potential analgesic drugs, and particularly forevaluating the contribution of the desensitization mechanisms to theantinociceptive effects of endogenous opioids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a linear model of the β-arrestin2/S65T-Green FluorescentProtein (GFP) conjugate.

FIG. 2A provides the results of a Western Blot of homogenates of HEK-293cells expressing the βarr2-GFP conjugate as well as endogenousβ-arrestin2. βarr2 indicates endogenous cellular β-arrestin2; βarr2-GFPindicates βarrestin2-GFP conjugate; approximate molecular weights areindicated to the right of the gel. Lane 1 was treated withanti-βarrestin antibody; Lane 2 with anti-GFP antibody.

FIG. 2B shows the sequestration of β2AR in COS cells with and withoutoverexpressed β-arrestin2 (left two bars) and with and withoutoverexpressed βarr2-GFP (right two bars). Wild type β-arrestin2 andβarr2-GFP enhanced β2AR sequestration equally well above control levels,producing a 2.5 and 2.4 fold increase, respectively.

FIG. 3A: Confocal microscopy photomicrographs show βarr2-GFPtranslocation from cytosol (panel 1 at left) to membrane (panel 2 atright) in HEK-293 cells containing the β2AR, due to the addition of theβAR2 agonist isoproterenol. Bar=10 microns.

FIG. 3B: Confocal microscopy photomicrographs show βarr2-GFPtranslocation from cytosol (panel 1 at left) to membrane (panel 2 atright) in COS cells containing the β2AR, and due to addition of the βAR2agonist isoproterenol. Bar=10 microns.

FIG. 4 depicts a HEK-293 cell containing 12CA5(HA) tagged β2AR (confocalmicroscopic photographs). FIG. 4A shows a cell after reorganization ofβ2AR into plasma membrane clusters. FIG. 4B provides three pictures ofthe same cell at 0, 3, and 10 minutes (left to right) after the additionof agonist. Redistribution of βarr2-GFP to the cell membrane is shown bythe enhancement of membrane fluorescence with a concomitant loss ofcytosolic fluorescence. Arrows indicate areas of co-localization; bar=10microns.

FIG. 5 shows the influence of overexpressed GRK on the redistribution ofβarr2-GFP in HEK-293 cells expressing the Y326A phosphorylation-impairedβ2AR. Cells without (Row A) and with (Row B) overexpressed GRKs wereexposed to agonist, and the real-time redistribution of βarr2-GFP wasobserved. βarr2-GFP translocation in cells containing overexpressed GRK(Row B) was more robust, indicating an increased affinity of βarr2-GFPfor receptor. Bar=10 microns.

FIG. 6A depicts the agonist-induced time dependent translocation ofβarr2-GFP to beta2 adrenergic receptors in a representational HEK-293cell.

FIG. 6B graphs the time course of agonist-induced translocation ofβarr2-GFP to beta2 adrenergic receptors in HEK-293 cells; this graph isquantitative and is based on the responses of a plurality of cells.

FIG. 6C is depicts the agonist-induced translocation of βarr2-GFP tobeta2 adrenergic receptors in representational HEK-293 cells, at varyingdoses of agonist.

FIG. 6D graphs the dose dependent agonist-induced translocation ofβarr2-GFP to beta2 adrenergic receptors in HEK-293 cells; this graph isquantitative and is based on the responses of a plurality of cells.

FIG. 6E evaluates the translocation of βarr2-GFP from the cell cytosolto the cell membrane, in response to exposure to receptor agonist(middle panel) and subsequent exposure to receptor antagonist (rightpanel).

FIG. 7 illustrates characteristics of the targeted disruption of themouse βarrestin-2 (βarr2) gene.

FIG. 7A depicts schematic diagrams of βarr2 gene (top), targeting vector(middle) and the homologous recombinant gene (bottom) (7). The arrowsindicate the translational start and stop sites. The black boxesindicate the exons. A 0.8 kb Bam HI-Hind III fragment was replaced withthe pGK-neo cassette such that the entire exon 2, encoding amino acids9-19, was deleted. Transcription of the neomycinresistant gene opposedthat of the βarr2 gene. Both 5′ and 3′ external probes were used ingenotype screening. Restriction enzyme sites are as follows: B, Bam HI;N, Nco 1; H, Hind III; R, Eco RI.

FIG. 7B illustrates southern blot analysis of genomic DNA from wild type(WT), heterozygous (+/−) and homozygous (−/−) mice. Tail DNA wasdigested with Bam HI and analyzed by Southern blotting with the 5′ probeas shown in (A). A 3.5-kb fragment is indicative of the βarr2 knock-out(KO) allele and a 3-kb fragment is indicative of the wild-type allele.

FIG. 7C depicts protein immunoblot analysis of βarr2 expression in WT,βarr2 and βarr2-KO mice. Membranes were blotted for βarr (top) and βarr2(bottom) protein expression. Each lane was loaded with 25 pLg proteinderived from the same lysates of the indicated brain regions.

FIG. 8 illustrates enhanced and prolonged morphine-inducedantinociception in βarr2-KO mice. Antinociceptive responses weremeasured as hot plate (56° C.) response latency after morphine (10mg/kg, s.c.) treatment. The “response” was defined by the animal eitherlicking the fore- or hind-paws or flicking the hind-paws. In thesestudies, the most prominent response was fore-paw licking. To avoidtissue damage the animals were not exposed to the plate for more than 30seconds. Data are reported as the percent of the maximal possibleresponse time (30 seconds) which was determined from each individualmouse's basal response, the response after drug treatment, and theimposed maximum cutoff time with the following calculation (F. Porrecaet al., J Pharmacol Exp Ther 230, 341 (1984); J. Belknap et al., PhysiolBehav 46, 69 (1989). M. Gardmark et al., Pharmacol Toxicol 83, 252(1998); G. Elmer et al., Pain 75, 129 (1998)): 100% ×[(Drug responsetime-Basal response time)/(30 sec-Basal response time)]=% maximumpossible effect (% MPE). WT (n=6), heterozygotes (+/−, n=5) and KO (n=9)mice were analyzed together in the same experiment. The % MPE curves ofthe βarr2-KO and βarr2+/−mice were significantly greater than the WTresponse curve (P<0.001) as determined by two-way ANOVA.

FIG. 9 depicts greater dose-dependent antinociceptive responses tomorphine in βarr2-KO mice. The degree of antinociception was determinedby measuring latency of hot plate (56° C.) responses (FIG. 2).Withdrawal latencies were measured 30 min. after a first dose ofmorphine (1 mg/kg, s.c.) at which point, animals were immediatelyinjected with 4 mg/kg, s.c. morphine for a cumulative dose of 5 mg/kg.Antinociception was again assessed after 30 min. and mice wereimmediately injected with morphine (5 mg/kg, s.c.), to give a finalcumulative dose of 10 mg/kg. Withdrawal latencies were again measuredafter 30 min. after which, mice were immediately injected with naloxone(2.5 mg/kg, s.c.). After 10 min., antinociception was assessed oncemore. WT (n=7) and βarr2-KO (n=6) mice were significantly different ateach dose (*P<0.01, **P<0.001; Student's t-test). Means±S.E.M. areshown. In an additional experiment, morphine (25 mg/kg, s.c.) inducedthe maximum imposed response (100%) in both genotypes. Thus, anapproximate 2 fold shift in the apparent ED₅₀ values was observedbetween genotypes [WT: 9.77 (8.08-11.81) mg/kg; KO: 5.98 (5.10-6.94)mg/kg (95% confidence intervals)].

FIG. 10 depicts increased hypothermic responses to morphine in βarr2-KOmice. Rectal body temperatures were measured with a digital thermometer(M. Adler et al., Annu Rev Pharmacol Toxicol 28, 429 (1988); F.Fumagalli et al., J Neurosci 18, 4861 (1998) (TH8, Physitemp, Clifton,N.J., USA). The probe was inserted into the rectum and maintained untilthe temperature reading stabilized. Basal body temperatures did not varysignificantly between genotypes (WT: 36.4±0.1° C.; KO: 36.8±0.1° C.).VVT (n=5) and KO (n=5) animals were analyzed in parallel during the sameexperiment. The curves are significantly different (P<0.001) asdetermined by 2-way ANOVA. Means±S.E.M. are shown.

FIG. 11 illustrates binding of [³⁵S]GTPγS to periaqueductal graymembranes from βarr2-KO and wild type (WT) mice. [³⁵S]GTPγS binding toisolated periaqueductal gray (PAG) membranes (prepared as described inconjunction with Table 1 below) was determined after 2 hour stimulation(30° C.) with 50-10,000 nM of the mOR-selective agonist, [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO). PAG membranes (10 μg protein perassay tube) were incubated in the presence of 10 μM GDP and 50 pM[³⁵S]GTPγS (1250 Ci/mmol, NEN, Boston, Mass.). [³⁵S]GTPγS binding wasmeasured as described (P. Portoghese, in Handbook of ExperimentalPharmacology: Opioids 1, A. Herz, Ed. (Springer-Verlag, New York, 1993)p.p. 279293. A. et al., ibid, p.p. 645-679). [³⁵S]GTPγS binding isexpressed as percent increase in [³⁵S]GTPγS binding relative to bindingin unstimulated samples. Data were analyzed by nonlinear regressionusing GraphPad Prism software and are presented as the mean±S.E.M of atleast three experiments performed in triplicate wherein WT and βarr2-KObrain regions were assayed simultaneously. In the absence of agoniststimulation, basal [³⁵S]GTPγS binding was: WT: 440±83 cpm and βarr2-KO:527±99 cpm.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “arrestin ”as used herein has its ordinary meaning in the artand is intended to encompass all types of arrestin, including but notlimited to visual arrestin (sometimes referred to as Arrestin 1),βarrestin 1 (sometimes referred to as Arrestin 2), and βarrestin 2(sometimes referred to as Arrestin 3).

The term “βarrestin” (or “βarrestin”) as used herein is intended toencompass all types of βarrestin, including but not limited to βarrestin1 and βarrestin 2.

The phrases “concurrent administration,” “administration in combination,if “simultaneous administration” or “administered simultaneously” asused herein, interchangeably mean that the compounds are administered atthe same point in time or immediately following one another. In thelatter case, the two compounds are administered at times sufficientlyclose that the results observed are indistinguishable from thoseachieved when the compounds are administered at the same point in time.

As used herein, exogenous or heterologous DNA (or RNA) refers to DNA (orRNA) which has been introduced into a cell (or the cell's ancestor)through the efforts of humans. Such heterologous DNA may be a copy of asequence which is naturally found in the cell being transformed, or asequence which is not naturally found in the cell being transformed, orfragments thereof.

As used herein, the term ‘gene’ refers to a DNA sequence thatincorporates (1) upstream (5′) regulatory signals including a promoter,(2) a coding region specifying the product, protein or RNA of the gene,(3) downstream (3′) regions including transcription termination andpolyadenylation signals and (4) associated sequences required forefficient and specific expression.

Use of the phrase “substantial sequence similarity” in the presentspecification refers to DNA, RNA or amino acid sequences which haveslight and non-consequential sequence variations from a sequence ofinterest, and are considered to be equivalent to the sequence ofinterest. In this regard, “slight and non-consequential sequencevariations” mean that “similar” sequences (i.e., sequences that havesubstantial sequence similarity) will be functionally equivalent.Functionally equivalent sequences will function in substantially thesame manner to produce substantially the same compositions.

As used herein, a “native DNA sequence” or “natural DNA sequence” meansa DNA sequence which can be isolated from non-transgenic cells ortissue. Native DNA sequences are those which have not been artificiallyaltered, such as by site-directed mutagenesis. Once native DNA sequencesare identified, DNA molecules having native DNA sequences may bechemically synthesized or produced using recombinant DNA procedures asare known in the art.

As used herein, “a regulatory element” from a gene is the DNA sequencewhich is necessary for the expression of the gene, such as a promoter.In this invention, the term “operatively linked” to means that followingsuch a link a regulatory element can direct the expression of a linkedDNA sequence.

The term ‘promoter’ refers to a region of a DNA sequence thatincorporates the necessary signals for the efficient expression of acoding sequence. This may include sequences to which an RNA polymerasebinds but is not limited to such sequences and may include regions towhich other regulatory proteins bind together with regions involved inthe control of protein translation and may include coding sequences.Suitable promoters will be apparent to those skilled in the art, andwill vary depending upon the cell in which the DNA is to be expressed. Asuitable promoter for use in DNA constructs encoding aβarrestin/detectable molecule construct may be a promoter naturallyfound in the cell in which expression is desired; optionally, thepromoter of the βarrestin within the construct may be utilized. Bothinducible and constitutive promoters are contemplated for use in thepresent invention.

DNA Constructs

DNA constructs, or “expression cassettes,” of the present inventioninclude, 5′ to 3′ in the direction of transcription, a promoter, a DNAsequence operatively associated with the promoter, and, optionally, atermination sequence including stop signal for RNA polymerase and apolyadenylation signal for polyadenylase. All of these regulatoryregions should be capable of operating in the cell to be transformed.Suitable termination signals for a given DNA construct will be apparentto those of skill in the art.

The term “operatively associated,” as used herein, refers to DNAsequences on a single DNA molecule which are associated so that thefunction of one is affected by the other. Thus, a promoter isoperatively associated with a DNA when it is capable of affecting thetranscription of that DNA (i.e., the DNA is under the transcriptionalcontrol of the promoter). The promoter is said to be “upstream” from theDNA, which is in turn said to be “downstream” from the promoter.

The present inventors have determined that βarrestin redistribution fromthe cytosol to the plasma membrane occurs in response to agonistactivation of GPCRs. The present inventors demonstrated a common rolefor βarrestin in agonist-mediated signal transduction terminationfollowing agonist activation of receptors. The present inventors havedevised convenient methods of assaying agonist stimulation of GPCRS invivo and in vitro in real time. Although the pharmacology of members ofthe GPCR superfamily differs, the methods of the present inventionutilize βarrestin translocation to provide a single-step, real-timeassessment of GPCR function for multiple, distinct members of the GPCRsuperfamily. The present methods may additionally be utilized instudying and understanding the mechanisms of actions of varioustherapeutic agents. The present inventors have determined that a proteinconjugate or chimera comprising an arrestin molecule and a detectablemolecule (such as Green Fluorescent Protein) is useful in such methodsof assaying in vivo GPCR activity.

Due to the therapeutic importance of GPCRs, methods for the rapidscreening of compounds for GPCR ligand activity are desirable.Additionally, methods of screening orphan GPCRs for interactions withknown and putative GPCR ligands assist in characterizing such receptors.Optical methods are available for studying labelled protein dynamics inintact cells, including video microscopy, fluorescence recovery afterphotobleaching, and resonance energy transfer. However, such methods areof limited usefulness in labeling GPCRs for study, due to the relativelylow level of GPCR expression and the alterations in receptor functionthat can occur after tagging or labeling of the receptor protein.Radiolabeling or fluorescent labeling of test ligands has also beenutilized in screening for GPCR ligands. See, e.g. Atlas et al., Proc.Natl. Acad. Sci. USA 74:5490 (1977); U.S. Pat. No. 5,576,436 to McCabeet al. (all patents cited herein are incorporated herein in theirentirety). The introduction of foreign epitopes into receptor cDNA toproduce hybrid GPCRs is now a standard technique, and enhances detectionof GPCRs by monoclonal antibody technology. However, such techniques arelimited in their applicability to living cells. U.S. Pat. No. 5,284,746to Sledziewski describes yeast-mammalian hybrid GPCRs and methods ofscreening for GPCR ligands using such hybrid receptors. U.S. Pat. No.5,482,835 to King et al. describes methods of testing in yeast cells forligands of mammalian GPCRs. However, application of these techniques tothe study or identification of orphan GPCRs requires prior knowledge ofligands or signal transduction events and are therefor not generallyapplicable or universal.

Phosphorylation of GPCRs is a mechanism leading to desensitization ofthe receptors; receptors that have been continuously or repeatedlystimulated lose responsiveness, whereas the responses of other receptorsremain intact. See Harden, Pharmacol. Rev. 35:5 (1983); Benovic et al.,Annu. Rev. Cell. Biol. 4:405(1988). In a variety of cells, specifickinases have evolved for specific GPCRs. Desensitization occurs via thefollowing pathway: agonist occupancy of the receptor transforms thereceptor into an appropriate substrate for an associated kinase;βarrestin binds to the kinase phosphorylated receptor and preventssubsequent interaction with the appropriate G-protein, as well asinitiating both internalization and resensitization processes. Fergusonet al, Science, 271:363 (1996); Lohse et al., Science 248:1547 (1990).βarrestin dependent desensitization is induced only when the GPCR isactivated by ligand binding, and is an example of homologousdesensitization (i.e., the ligand desensitizes only its targetreceptors). Lohse et al. (1990) and Attramadal et al., J. Biol. Chem.267:17882 (1992) provide cDNA and amino acid sequences of βarrestin.Various isoforms of βarrestin are known; as used herein, βarrestinrefers to all such isoforms of βarrestin, proteins having substantialsequence similarity thereto which are functional βarrestins, andfunctional fragments thereof. Functional fragments of βarrestin, itsisoforms and analogs, may be determined using techniques as known in theart.

Molecules detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical and optical means are known. Opticallydetectable molecules include fluorescent labels, such as commerciallyavailable fluorescein and Texas Red. Detectable molecules useful in thepresent invention include any biologically compatible molecule which maybe conjugated to a βarrestin protein without compromising the ability ofβarrestin to interact with the GPCR system, and without compromising theability of the detectable molecule to be detected. Conjugated molecules(or conjugates) of βarrestin and detectable molecules (which also may betermed ‘detectably labelled βarrestins’) are thus useful in the presentinvention. Preferred are detectable molecules capable of beingsynthesized in the cell to be studied (e.g., where the cell can betransformed with heterologous DNA so that the βarrestin-detectablemolecule chimera is produced within the cell). Particularly preferredare those detectable molecules which are inherently fluorescent in vivo.Suitable detectable molecules must be able to be detected withsufficient resolution within a cell that translocation of βarrestin fromthe cytosol to the cell membrane in response to agonist binding to GPCRcan be qualitatively or quantitatively assessed. Molecules detectable byoptical means are presently preferred.

Fusion proteins with coding sequences for β-galactosidase, fireflyluciferase, and bacterial luciferase have been used in methods ofdetecting gene expression and protein interactions in cells. However,these methods require exogenously-added substrates or cofactors. In themethods of the present invention, an inherently fluorescent markermolecule is preferred, such as GFP, since detection of such a markerintracellularly requires only the radiation by the appropriatewavelength of light and is not substrate limited.

Green Fluorescent Protein (GFP) was first isolated from the jelly fishAequorea victoria, and has an inherent green bioluminescence that can beexcited optically by blue light or nonradiative energy transfer.Sequences of GFP-encoding cDNA and GFP proteins are known; see, e.g.,Prasher et al., Gene, 111:229 (1992). The crystalline structure of GFPis described in Ormo et al., Science 273:1392 (1996). Purified nativeGFP absorbs blue light (maximally at 395 nm with aminor peak at 470 m)and emits green light (peak emission at 509 nm) (Morise et al,Biochemistry, 13:2656 (1974); Ward et al., Photochem. Photobiol., 31:611(1980)). It has been shown that GFP expressed in prokaryotic andeukaryotic cells produces a strong green fluorescence when excited bynear UV or blue light (see U.S. Pat. No. 5,491,084 to Chalfie andPrasher); as this fluorescence requires no additional gene products fromA. victoria, chromophore formation is not species specific and occurseither through the uses of ubiquitous cellular components or byautocatalysis. Expression of GFP in Escherichia coli results in aneasily detected green fluorescence that is not seen in control bacteria.See Chalfie et al., Science 263:802 (1994); U.S. Pat. No. 5,491,084.Cells expressing the green-fluorescent proteins may be convenientlyseparated from those which do not express the protein by afluorescence-activated cell sorter.

As used herein, Green Fluorescent Protein refers to the variousnaturally occurring forms of GFP which can be isolated from naturalsources, as well as artificially modified GFPs which retain thefluorescent abilities of native GFP. As discussed in Ormo et al.,Science 273:1392 (1996), various mutants of GFP have been created withaltered excitation and emission maxima. Two characteristics of wild-typeGFP which affect its usefulness in mammalian cell lines are the need toexcite it at UV wavelengths to obtain a maximal fluorescent signal, anddecreased fluorescence at temperatures over 23.degree. C. However, theS65T/GFP mutant overcomes these limitations. Heim et al., Proc. Natl.Acad. Sci. USA 91:12501 (1994). Additional alterations in the GFPprotein sequence which provide inherently fluorescent, biologicallycompatible molecules will be apparent to those in the art; sequencealterations may be made to alter the solubility characteristics of theprotein, its excitation wavelength, or other characteristics, whileretaining useful fluorescent properties. See, e.g. U.S. Pat. No.5,625,048 to Tsien and Heim; WO 9711091 (Bjorn, Poulsen, Thastrup andTullin); WO 9627675 (Haseloff, Hodge, Prasher and Siemering); WO 9627027(Ward); WO 9623898 (Bjorn et al.); WO 9623810 (Heim and Tsien); WO9521191 (Chalfie and Ward).

Cells useful in the methods of the present invention include eukaryoticand prokaryotic cells, including but not limited to bacterial cells,yeast cells, fungal cells, insect cells, nematode cells, plant or animalcells. Suitable animal cells include, but are not limited to HEK cells,HeLa cells, COS cells, and various primary mammalian cells. Cellscontained in intact animals, including but not limited to nematodes,zebrafish (and other transparent or semi-transparent animals) andfruitflies, may also be used in the methods of the present invention. Ananimal model expressing a βarrestin-detectable molecule fusion proteinthroughout its tissues, or within a particular organ or tissue type,will be useful in studying cellular targets of known or unknown GPCRligands.

Cells useful in the present methods include those which express a knownGPCR or a variety of known GPCRs, or which express an unknown GPCR or avariety of unknown GPCRs. As used herein, a cell which expresses a GPCRis one which contains that GPCR as a functional receptor in itsmembrane; the cells may naturally express the GPCR(s) of interest, ormay be genetically engineered to express the GPCR(s) of interest. Asused herein, an ‘unknown’ or ‘orphan’ receptor is one whose function isunknown, and/or whose ligands are unknown.

The Present Experiments

Green fluorescent protein (GFP) has been used to study protein-proteininteractions in living cells. See Kaether & Gerdes, FEBS Lett. 369:267(1995); Olson et al., J. Cell. Biol. 130:639 (1995). Green fluorescentprotein (GFP) is useful as a reporter molecule for fusion proteins dueto its inherent fluorescence and its folding, which apparently isolatesit from its conjugated partner. Prasher et al., Gene 111:229 (1992);Ormo et al., Science 273:1392 (1996). For example, a seven transmembraneprotein as complex as the β2AR, which is three times larger than GFP,exhibits normal biochemistry after GFP conjugation to its C-terminus.Barak et al., Mol. Pharmacol. 51:177 (1997).

The present inventors established that a fusion protein consisting of aβarrestin molecule (βarrestin2) conjugated to a GFP at its C-terminus(βarr2-GFP, FIG. 1) is expressed in cells and is biologically active.The βarr2-GFP fusion protein is approximately 50% larger thanβarrestin2, and this size increase is reflected by its slower migrationon SDS-Page (FIG. 2A). The left lane of FIG. 2A, exposed to an antibodyagainst βarrestin, shows that βarr2-GFP runs more slowly than endogenousβarrestin2 (highlighted middle band). The right lane of FIG. 2A, treatedwith a monoclonal anti-GFP antibody, demonstrates that the slower banddoes indeed contain GFP. β2AR normally sequesters poorly in COS cells,and this has been correlated to the relatively poor expression ofendogenous βarrestins in COS cells. Menard et al., Mol. Pharmacol.51:800 (1997); Zhang et al., J. Biol. Chem. 271:18302 (1996).Overexpression of exogenous βarrestin enhances β2AR sequestration inthese cells; similarly, as shown herein, βarr2-GFP overexpression in COScells augmented β2AR internalization (FIG. 2B), demonstrating thatβarr2-GFP is biologically active and equivalent to native βarrestin.

Biochemical evidence indicates that βarrestins are predominantlycytosolic proteins. Ferguson et al., Can. J. Physiol. Pharmacol. 74:1095(1996). The present inventors, using confocal microscopy of βarr2-GFP inHEK-293 cells (FIG. 3A, left panel), confirmed that βarr2-GFP isdistributed throughout the cytosol and excluded from the nucleus. Thepresent data also establish for the first time that βarrestin is notpredominantly compartmentalized at the plasma membrane in the absence ofagonist but that, upon addition of saturating concentrations of anagonist to the cell medium, βarrestin is translocated from cell cytosolto cell membrane. Where βarrestin is conjugated to an opticallydetectable molecule such as GFP, as shown herein, a rapid and readilyobservable optical enhancement of the membrane and a concomitant loss ofcytosolic optical signals occurs (see FIGS. 3A and 3B, where membranefluorescence is enhanced and cytosol fluorescence is decreased due totranslocation of the βarrestin-GFP chimera).

To investigate whether the intracellular translocation of βarrestintargeted binding sites in the plasma membrane other than the β2AR, thepresent inventors first crosslinked the receptors using monoclonalantibodies. As reported herein and shown in FIG. 4, the geometry of theagonist-induced time dependent translocation of βarrestin to the plasmamembrane mimicked the distribution of pre-aggregated β2ARs, indicatingthat the targeted site of βarrestin is indeed β2AR or an associatedcomponent.

It has been postulated that phosphorylation of GPCRs by GRKs facilitatesdesensitization by increasing their affinity for βarrestins. Gurevich etal, J. Biol. Chem. 268:16879 (1993); Gurevich et al., J. Biol. Chem.268:11628 (1993). When expressed in HEK-293 cells and exposed toagonist, mutant Y326A-β2ARs are not significantly phosphorylated byendogenous GRKs (Ferguson et al., J. Biol. Chem., 270:24782 (1995).Therefore, the present inventors utilized this mutant receptor toinvestigate the above question of βarrestin affinity in vivo. Y326A-β2ARwas cotransfected with βarr2-GFP into HEK cells in the absence andpresence of co-transfected GRK. If the above hypothesis were true,reversal of phosphorylation impairment by overexpressed GRKs wouldresult in a noticeable difference in βarr2-GFP translocation. Asreported herein, without added GRK, βarr2-GFP translocation in responseto agonist proceeded poorly; with the addition of GRK, βarr2-GFPtranslocation to the plasma membrane was much more robust (FIG. 5),indicating the importance of phosphorylation to βarrestin activity.

The present inventors determined that translocation of βarrestin fromthe cell cytosol to the cell membrane is an indicator of agoniststimulation of GPCR activity, and that a chimeric protein comprisingβarrestin and the detectable molecule GFP was capable of detectablydisplaying the real-time translocation of βarrestin in response toagonist activation of GPCRs.

The results presented herein establish that βarrestin targets GPCRs oran associated molecule following agonist binding and receptorphosphorylation. These data demonstrate a biological behavior forβarrestin that has only been postulated from biochemical studies, andcharacterize for the first time how βarrestin compartmentalizationchanges after initiation of receptor signal transduction. Agonistactivation of a GPCR ultimately culminates in the association ofβarrestins with GPCRs, thus the visualization of the agonist mediatedβarrestin translocation process provides a universal indicator of GPCRactivation.

The present inventors have demonstrated that GPCR signal transductioninduces a rapid, substantial increase in the relative and absoluteamount of plasma membrane bound βarrestin. The agonist-mediatedredistribution of βarrestin coupled to a detectable molecule provides anoptical amplification of the extracellular signals transduced by GPCRs,and this occurs simultaneous with, or within the same time frame as, thechemical amplification normally provided by second messenger cascades.Chimeras of βarrestin and a detectable molecule are useful for the studyof βarrestin kinetics and GPCR related behavior such as endocytosis.Additionally, such chimeras are useful as biosensors for signaling whenGPCRs become activated, and provide methods of screening compounds forGPCR activity, and screening orphan GPCRs for ligand responsiveness. Inaddition, the ability of co-transfected GRKs to enhance both the rateand extent of βarrestin translocation indicate that the present methodsand constructs can also be used to monitor GRK activity, as well asmonitor drugs, proteins and compounds for activation or inhibition ofthe GRK/βarrestin process.

The present invention provides a method for screening compounds for GPCRagonist activity, comprising: a) providing a cell expressing a known orunknown GPCR and containing a chimeric protein comprising a βarrestinprotein and a visually detectable protein; b) exposing the cell to atest compound; and c) detecting translocation of the detectable moleculefrom the cytosol of the cell to the membrane edge of the cell; wheretranslocation of the detectable molecule from the cytosol to themembrane edge of the cell indicates activation of the GPCR and,accordingly, the GPCR activating effect of the test compound.Translocation of the chimeric protein is evidenced by an increase in theintensity of detectable signal located at the membrane edge (and/or adecrease in the cytosol), where the change occurs after exposure to thetest compound. Translocation may thus be detected by comparing changesin the detectable signal in the same cell over time (i.e., pre and posttest compound exposure). Alternatively, a test cell may be compared to acontrol cell (no exposure to test compound), or a test cell may becompared to a pre-established standard. If a known agonist is availablethe present methods can be used to screen for and study GPCRantagonists. Additionally, the membrane association of βarrestin shouldbe increased by expression of an excess of receptor or by aconstitutively active GPCR that undergoes phosphorylation by GRKs evenin the absence of agonist. Therefore, the present methods can be used tomonitor for inverse agonists of GPCRs.

Methods of detecting the intracellular translocation of the chimericprotein will depend on the particular detectable protein utilized; oneskilled in the art will be able to readily devise detection methodssuitable for particular detectable molecules, given the teachings of thepresent specification and knowledge in the art. In a preferredembodiment, the visually detectable protein is a green-fluorescentprotein (GFP) as discussed below.

The methods of the present invention provide easily detectable results.The translocation of βarrestin coupled to a detectable molecule such asGFP, in response to GPCR activation, results in a relative enhancementof the detectable signal at the cell edge (i.e., at the cell membrane).In addition, the concomitant decrease in detectable signal from the cellcytosol means that ‘background noise’ (detectable signals which do notchange in response to GPCR activation) is minimized. In certain cells,activation of GPCRs will result in essential clearing of detectablesignal from the cytosol, and a 100-fold increase (or more) in thedetectable signal at the cell membrane. In the present methods, it ispreferred that the detectable signal at the membrane edge increase,after GPCR activation, at least two-fold, more preferably at least3-fold, and more preferably at least 5-fold or at least ten-fold.

As used herein, the introduction of a chimeric protein into a cell maybe accomplished by introducing into the cell (or the cell's ancestor) anucleic acid (e.g., DNA or RNA) sequence or construct encoding thechimeric protein, and culturing the cell in an environment which allowsexpression of the chimeric protein. Introduction of nucleic acidsencoding the chimeric protein, or introduction of the protein itself,into a cell may be carried out by any of the many suitable methods whichare known in the art, including transfection, electroporation,microinjection, and liposome delivery.

The present invention provides a DNA construct comprising a promoter,DNA encoding a βarrestin protein operatively associated therewith, andDNA encoding a visually detectable marker protein operatively associatedtherewith. The promoter is operatively associated with the encoding DNA;DNA encoding βarrestin may be 5′ from DNA encoding the visuallydetectable marker, or vice versa. In a preferred embodiment, the DNAencoding a visually detectable marker encodes a green-fluorescentprotein (GFP). Vectors comprising such DNA constructs are a furtheraspect of the present invention.

The present invention further provides conjugates (such as chimericproteins or fusion proteins) which comprise a βarrestin protein and avisually detectable protein. In a preferred embodiment, the visuallydetectable protein is a green-fluorescent protein (GFP).

The present invention further provides a cell comprising a DNA molecule,which DNA molecule comprises, in the 5′ to 3′ direction, a promoter, DNAencoding a βarrestin protein operatively associated therewith, and DNAencoding a visually detectable marker protein operatively associatedtherewith. In a preferred embodiment, the DNA encoding a visuallydetectable marker encodes a green-fluorescent protein (GFP).

The cells of the present invention may be used to detect the presence ofspecific molecules in various kinds of samples such as, e.g., aqueoussamples, biological samples (for example blood, urine or saliva),environmental samples, or industrial samples. In such uses, the cellscontain a GPCR whose agonists are known. Activation of the GPCR and theconcomitant translocation of the detectable signal from the cytosol tothe membrane edge indicates the presence of the agonist for the GPCR. Acell used in such a method may contain only a single type of known GPCR,or a variety of known GPCRs. Such detection will be useful for medicaland veterinary diagnostic purposes; industrial purposes; and screeningfor drugs or chemicals of abuse or biological toxins that affectGPCR-mediated signal transduction.

The cells of the present invention may be deposited on, affixed to,supported by, or immobilized on a substrate. The substrate may be of anysuitable material which is not harmful or detrimental to the livingcells deposited thereon, i.e., which is bio-compatible with the livingmaterial deposited thereon. The substrate may be rigid, semi-rigid orflexible; and may be opaque, transparent, or semi-transparent. The size,geometry and other physical characteristics of the substrate will bedictated by the intended use, as will be apparent to one skilled in theart. Suitable substrates include, but are not limited to, plastics,glass, ceramics, silica, biocompatible monomer and polymer compositions,semiconductor materials, fiber optic materials, polystyrene, membranes,sephadex, and bio-organic materials. Examples of biocompatible materialsare provided in U.S. Pat. Nos. 5,578,079; 5,575,997 and 5,582,834 toLeung and Clark; and U.S. Pat. No. 5,522,896 to Prescott.

The present invention further provides methods for screening for thepresence of a GPCR agonist in a solution which comprises: a) providing acell expressing a known or unknown GPCR and containing a chimericprotein comprising a βarrestin protein and a visually detectableprotein; b) exposing the cell to a test solution; and c) detectingtranslocation of the detectable molecule from the cytosol of the cell tothe membrane edge of the cell; where translocation of the detectablemolecule from the cytosol to the membrane edge of the cell indicatesactivation of the GPCR and, accordingly, the GPCR agonist effect of thetest solution. Translocation of the chimeric protein is evidenced asdiscussed above.

The present invention further provides methods for screening for thepresence of a GPCR antagonist in a solution which comprises: a)providing a cell expressing a GPCR and containing a chimeric proteincomprising a βarrestin protein and a visually detectable protein; b)exposing the cell to a test compound; then c) exposing the cell to aknown agonist to the GPCR expressed in the cell; and d) detectingtranslocation of the detectable molecule from the cytosol of the cell tothe membrane edge of the cell. If the test compound contains anantagonist, translocation of the detectable molecule will be delayed fora period of time corresponding to duration of antagonist action on thereceptor (which time period will vary depending on the antagonist and/orthe receptor). Translocation of the detectable molecule from the cytosolto the membrane edge of the cell indicates activation of the GPCR by theagonist. Accordingly, when translocation does not occur or is delayed(compared to that which would occur in the absence of test compound),the test compound contains an antagonist to the GPCR. Absence or delayof translocation may be assessed by comparison to a control cell (notexposed to test compound) or to a predetermined standard. Translocationof the chimeric protein is evidenced as discussed above. Exposure to thetest compound and the known agonist may occur at essentially the sametime, or exposure to the agonist may occur subsequent to exposure to thetest compound. As used herein, subsequent exposure refers to exposurewithin the time period during which a potential antagonist would beexpected to be interacting with the GPCR (i.e., binding to or bound tothe GPCR).

The present invention further provides methods for screening a cell forthe presence of a GPCR, comprising: a) providing a test cell; b)introducing into the test cell a chimeric protein comprising a βarrestinprotein and a visually detectable protein; and then c) exposing the cellto a test solution containing a known agonist to a GPCR; and d)detecting translocation of the detectable molecule from the cytosol ofthe cell to the membrane edge of the cell; where translocation of thedetectable molecule from the cytosol to the membrane edge of the cellindicates activation of a GPCR and, accordingly, that the test cellcontains such a GPCR. Translocation of the chimeric protein is evidencedas discussed above.

The present invention further provides methods for screening a cellpopulation for the presence of cells containing GPCRs, comprising: a)providing a population of test cells, said test cells containingchimeric proteins comprising a βarrestin protein and a visuallydetectable protein; and then b) exposing the cell population to a testsolution containing an agonist to a GPCR; and d) detecting those cellsin which translocation of the detectable molecule from the cytosol ofthe cell to the membrane edge of the cell occurs; where translocation ofthe detectable molecule from the cytosol to the membrane edge of a cellindicates activation of a GPCR and, accordingly, that the cell inquestion contains a GPCR. Translocation of the chimeric protein isevidenced as discussed above. Populations of cells to be screenedinclude a collection of individual cells, a tissue comprising aplurality of similar cells, an organ comprising a plurality of relatedcells, or an organism comprising a plurality of tissues and organs.

As used herein, ‘exposing’ a cell to a test compound or solution meansbringing the cell exterior in contact with the test compound orsolution. Where the test compound or solution is being screened for GPCRligand activity, exposure is carried out under conditions that wouldpermit binding of a GPCR ligand to a receptor expressed in that cell. Asused herein, ‘translocation’ of βarrestin refers to movement of theβarrestin molecule from one area of the cell to another.

The present methods may further be used to assess or study the effectsof any molecule in the GPCR pathway which exerts its effect upstream ofβarrestin binding (i.e., prior to βarrestin binding to thephosphorylated GPCR). Thus the present invention provides methods forassessing GPCR pathway functions in general. As used herein, the GPCRpathway refers to the series of events which starts with agonistactivation of a GPCR followed by desensitization of the receptor via Gprotein-coupled receptor kinase (GRK) phosphorylation and βarrestinbinding.

In a broad sense the present invention thus provides a method ofscreening test compounds and test conditions for the ability to affect(activate or inhibit, enhance or depress) a GPCR pathway, and providesmethods of assessing GPCR pathway function in a cell in general. In thepresent methods, the extent of translocation of βarrestin is indicatedby the degree of detectable changes in the cell; the extent of βarrestintranslocation is an indicator of the extent of GPCR pathway completion.The relative extent of translocation under varied test conditions may becompared, or a test condition may be compared to a control condition orto a predetermined standard.

For example, the specificity and effects of various kinases (includingthose known to interact with GPCR pathways and those not previouslyknown to interact with GPCRs) for a specific GPCR or a group of GPCRsmay be assessed by providing a test kinase to a test cell expressing aGPCR and containing a detectable βarrestin molecule, exposing the cellto a GPCR agonist, and assessing the translocation of detectableβarrestin from the cell cytosol to the cell membrane (see Example 7herein). Translocation of the βarrestin to the cell membrane indicatesthat the test kinase, in response to agonist occupancy of the receptor,is able to bind to and phosphorylate the receptor, so that βarrestinwill then bind to the kinase phosphorylated receptor and preventsubsequent interaction with the appropriate G-protein. In similar ways,the function of altered, recombinant or mutant kinases may be assessed;compounds may be screened for the ability to activate or inhibit theGPCR pathway, G protein-coupled receptor kinases, or βarrestin binding;and the function of G-proteins may be assessed. For example, thefollowing test conditions may be assessed using methods as describedherein: the effects of G-proteins (including natural, heterologous, orartificially altered G-proteins) within the test cell; exposure of thetest cell to known or putative GPCR ligands; and co-expression of asecond receptor in the test cell expressing a GPCR.

Still further, the present methods allow the screening of βarrestins(naturally occurring, artificially introduced, or altered, mutant orrecombinant) for the ability to bind to a phosphorylated GPCR. In suchmethods, the test βarrestin is conjugated to a detectable molecule suchas GFP, and is placed within a cell containing a GPCR. The cell isexposed to a known agonist of the GPCR, and translocation of thedetectable molecule from the cytosol of the cell to the membrane edge ofthe cell is detected. The translocation of the detectable moleculeindicates that the test βarrestin protein is able to bind to thephosphorylated GPCR. As in other methods of the present invention, thetranslocation may be compared to a control cell containing a knownβarrestin, or to a predetermined standard.

G Protein Coupled Receptors

GPCRs suitable for use in the present methods are those in which agonistbinding induces G protein-coupled receptor kinase (GRK) phosphorylation;translocation of arrestin from the cytosol of the cell to the cellmembrane subsequently occurs. As it is believed that virtually allmembers of the GPCR superfamily desensitize via this common mechanism,examples of suitable types of GPCRs include but are not limited to betaand alpha adrenergic receptors; GPCRs binding neurotransmitters (such asdopamine); GPCRs binding hormones; the class of odorant receptors(taste, smell and chemotactic receptors as found in nasal mucosa and thetongue, and on sperm, egg, immune system cells and blood cells); theclass of type II GPCRs including secretin, glucagon, and other digestivetract receptors; light-activated GPCRs (such as rhodopsin); and membersof the type III family of GPCRs which include but are not limited tometabotopic glutamate receptors and GABA.sub.B receptors. In addition tonaturally occurring GPCRs, GPCRs may be specifically engineered orcreated by random mutagenesis. Such non-naturally occurring GPCRs mayalso be utilized in and screened by the present methods. The presentmethods may be utilized with any membrane receptor protein in whichagonist binding results in the translocation of βarrestin. Suchreceptors include growth factors that signal through G proteins.

Automated Screening Methods

The methods of the present invention may be automated to provideconvenient, real time, high volume methods of screening compounds forGPCR ligand activity, or screening for the presence of a GPCR ligand ina test sample. Automated methods are designed to detect the change inconcentration of labelled βarrestin at the cell membrane and/or in thecytosol after exposure to GPCR agonist. The alteration of βarrestindistribution can be detected over time (i.e., comparing the same cellbefore and after exposure to a test sample), or by comparison to acontrol cell which is not exposed to the test sample, or by comparisonto pre-established indicia. Both qualitative assessments(positive/negative) and quantitative assessments (comparative degree oftranslocation) may be provided by the present automated methods, as willbe apparent to those skilled in the art.

It is thus a further object of the present invention to provide methodsand apparatus for automated screening of GPCR activity, by detecting thetranslocation of detectably labeled βarrestin from cell cytosol to cellmembrane in response to agonist activation of GPCRs. The translocationmay be indicated by an alteration in the distribution of a detectablesignal within a cell over time, between a test cell and a control cell,or by comparison to previously established parameters. In particular,according to one embodiment of the present invention, a plurality ofcells expressing GPCRs and containing chimeric proteins comprising adetectable molecule and a βarrestin molecule are provided. Indicia ofthe distribution of the detectable molecules are then measured usingconventional techniques. In various embodiments, (a) measurement ofoptical indicia occurs before and after the addition of a test sample toa cell, and the time point measurements are compared; (b) opticalindicia are measured in a test cell exposed to a test sample and in anon-exposed control cell, and these measurements are compared; and (c)measurement of a test cell after addition of a test sample is comparedto preestablished parameters. The optical indicia being measured may befluorescence signals (e.g., fluorescence intensities) if the detectablemolecule of the chimeric βarrestin protein is a fluorescent indicatorsuch as GFP. Other optical indicia that are suitable for real-timemeasurement may also be used, as will be apparent to those skilled inthe art.

An embodiment of the present invention includes an apparatus fordetermining GPCR response to a test sample. This apparatus comprisesmeans, such as a fluorescence measurement tool, for measuring indicia ofthe intracellular distribution of detectable βarrestin proteins in atleast one test cell, and optionally also in a control or calibrationcell. Measurement points may be over time, or among test and controlcells. A computer program product controls operation of the measuringmeans and performs numerical operations relating to the above-describedsteps. The preferred computer program product comprises a computerreadable storage medium having computer-readable program code meansembodied in the medium. Hardware suitable for use in such automatedapparatus will be apparent to those of skill in the art, and may includecomputer controllers, automated sample handlers, fluoresence measurementtools, printers and optical displays. The measurement tool may containone or more photodetectors for measuring the fluorescence signals fromsamples where fluorescently detectable molecules are utilized in thedetectable βarrestin construct. The measurement tool may also contain acomputer-controlled stepper motor so that each control and/or testsample can be arranged as an array of samples and automatically andrepeatedly positioned opposite a photodetector during the step ofmeasuring fluorescence intensity.

The measurement tool is preferably operatively coupled to a generalpurpose or application specific computer controller. The controllerpreferably comprises a computer program product for controllingoperation of the measurement tool and performing numerical operationsrelating to the above-described steps. The controller may accept set-upand other related data via a file, disk input or data bus. A display andprinter may also be provided to visually display the operationsperformed by the controller. It will be understood by those having skillin the art that the functions performed by the controller may berealized in whole or in part as software modules running on a generalpurpose computer system. Alternatively, a dedicated stand-alone systemwith application specific integrated circuits for performing the abovedescribed functions and operations may be provided.

As provided above, the indicia of βarrestin distribution may take theform of fluorescent signals, although those skilled in the art willappreciate that other indicia are known and may be used in the practiceof the present invention, such as may be provided by labels that producesignals detectable by fluorescence, radioactivity, colorimetry, X-raydiffraction or absorption or magnetism. Such labels include, forexample, fluorophores, chromophores, radioactive isotopes (e.g., ³²P or¹²⁵I) and electron-dense reagents.

The expression or transcription cassette may be provided in a DNAconstruct which also has at least one replication system. Forconvenience, it is common to have a replication system functional inEscherichia coli, such as Co1E1, pSC101, pACYC184, or the like. In thismanner, at each stage after each manipulation, the resulting constructmay be cloned, sequenced, and the correctness of the manipulationdetermined. In addition, or in place of the E. coli replication system,a broad host range replication system may be employed, such as thereplication systems of the P-1 incompatibility plasmids, e.g., pRK290.In addition to the replication system, there will frequently be at leastone marker present, which may be useful in one or more hosts, ordifferent markers for individual hosts. That is, one marker may beemployed for selection in a prokaryotic host, while another marker maybe employed for selection in a eukaryotic host. The markers may beprotection against a biocide, such as antibiotics, toxins, heavy metals,or the like; may provide complementation, by imparting prototrophy to anauxotrophic host; or may provide a visible phenotype through theproduction of a novel compound in the plant.

The various fragments comprising the various constructs, expressioncassettes, markers, and the like may be introduced consecutively byrestriction enzyme cleavage of an appropriate replication system, andinsertion of the particular construct or fragment into the availablesite. After ligation and cloning the DNA construct may be isolated forfurther manipulation. All of these techniques are amply exemplified inthe literature as exemplified by J. Sambrook et al., Molecular Cloning.A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory).

Arrestin Knockout Mice

The production of βarrestin knockout mice can be carried out in view ofthe disclosure provided herein and in light of techniques known to thoseskilled in the art, such as described in U.S. Pat. Nos. 5,767,337 toRoses et al.; 5,569,827 to Kessous-Elbaz et al.; and 5,569,824 toDonehower et al. (the disclosures of which applicants specificallyintend to be incorporated by reference herein in their entirety); and A.Harada et al., Nature 369, 488 (1994). Particularly preferred mice forcarrying out the present invention are also disclosed below.

1. Assay techniques. The step of determining whether or not βarrestinbinding to the phosphorylated μ opioid receptor is inhibited by the testcompound may be carried out by any suitable technique, including invitro assay and in vivo assay (e.g., in a cell that contains theβarrestin and the phosphorylated μ opioid receptor). A particularlysuitable technique for in vivo assay is as described previously. Ingeneral, this technique involves providing a cell that expresses μopioid receptor as a G-protein coupled receptor, and contains theβarrestin protein conjugated to an optically detectable molecule (e.g.,green fluorescent protein). The test compound is then introduced intothe cell (e.g., by microinjection, by electroporation, by suspending thecell in an aqueous solution that contains the test compound, bycontacting the cell to liposomes that contain the test compound, byinsertion of a heterologous nucleic acid into the cell that encodes andexpresses the test compound, etc.). Translocation of the molecule fromthe cytosol of the cell to the membrane edge of the cell is thenmonitored or examined, with the inhibition of such translocationindicating that the test compound inhibits the binding of βarrestin tothe phosphorylated μ opioid receptor. If desired, phosphorylation of theμ opioid receptor can be induced or enhanced by any suitable means, suchas contacting a μ opioid receptor agonist such as morphine to the cellin an amount effective to induce phosphorylation (e.g., by adding theagonist to the culture medium or liquid medium in which the cell iscontained). The cell is preferably a mammalian cell, but any suitablecell can be employed, including bacterial cells, yeast cells, fungalcells, plant cells, and other animal cells, so long as they express μopioid receptor and phosphorylate, or can be induced to phosphorylate,the same, and contain the desired βarrestin protein coupled to anoptically detectable molecule (e.g, either by exogenous introduction orexpression of the βarrestin conjugate therein). Any suitable βarrestinmay be employed as described above, with βarrestin-2 being preferred.

2. Test compounds. The present invention can be used with test compounds(or “probe molecules”), or libraries (where groups of different probemolecules are employed), of any type. In general, such probe moleculesare organic compounds, including but not limited to oligomers,non-oligomers, or combinations thereof. Nonoligomers include a widevariety of organic molecules, such as heterocyclics, aromatics,alicyclics, aliphatics and combinations thereof, comprising steroids,antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids,opioids, benzodiazepenes, terpenes, porphyrins, toxins, catalysts, aswell as combinations thereof. Oligomers include peptides (that is,oligopeptides) and proteins, oligonucleotides (the term oligonucleotidealso referred to simply as “nucleotide”, herein) such as DNA and RNA,oligosaccharides, polylipids, polyesters, polyamides, polyurethanes,polyureas, polyethers, poly (phosphorus derivatives) such as phosphates,phosphonates, phosphoramides, phosphonamides, phosphites,phosphinamides, etc., poly (sulfur derivatives) such as sulfones,sulfonates, suffites, sulfonamides, sulfenamides, etc., where for thephosphorous and sulfur derivatives the indicated heteroatom for the mostpart will be bonded to C, H, N, O or S, and combinations thereof.Numerous methods of synthesizing or applying such probe molecules onsolid supports (where the probe molecule may be either covalently ornon-covalently bound to the solid support) are known, and such probemolecules can be made in accordance with procedures known to thoseskilled in the art. See, e.g., U.S. Pat. No. 5,565,324 to Still et al.,U.S. Pat. No. 5,284,514 to Ellman et al., U.S. Pat. No. 5,445,934 toFodor et al. (the disclosures of all United States patents cited hereinare to be incorporated herein by reference in their entirety).

3. Pain control and active compounds. As noted above, the presentinvention provides a method of controlling pain in a subject, comprisinginhibiting βarrestin binding to the phosphorylated μ opioid receptor insaid subject in an amount effective to induce or enhance analgesia inthe subject. The method may be carried out with or without concurrentlyadministering a μ opioid receptor agonist such as morphine (or otheropiate, as described below). When carried out without concurrentadministration of μ opioid receptor, the analgesic activity relies uponthe activity of endogenous opioid receptor agonists.

The inhibiting of βarrestin binding (preferably βarrestin-2 binding) tophosphorylated μ opioid receptor can be carried out directly orindirectly by any suitable means, including but not limited to knockoutof the βarrestin gene as described herein, disabling or downregulatingthe kinase responsible for phosphorylation of the μ opioid receptor,administration of an antisense oligonucleotide that downregulatesexpression of the βarrestin, or the administration of an active compoundthat competitively inhibits binding of the βarrestin to phosphorylated μopioid receptor (which may be identified by the assay techniquesdescribed above). Obviously, functional μ opioid receptor itself mustremain in the cells (particularly nerve cells) of the subject so thatthe primary analgesic activity of the μ opioid receptor agonist can beexerted.

Compounds produced or identified as active compounds by application ofthe assay procedures described herein to the test compounds or probemolecules described herein are useful in vitro and in vivo as μ opioidreceptor agonists (in that they enhance the activity of opioids,although they do not bind to the same site as an opioid), are useful inenhancing the efficacy, potency, or analgesic activity of μ opioidreceptor agonists. Such compounds are also useful in vivo in controllingpain in a subject in need thereof. By “controlling pain”, “control ofpain” and the like herein is meant partially or completely inhibiting apain response or perception of pain in a subject, and/or partially orfully inducing local or general analgesia in a subject, either alone orin combination with another active agent administered to the subjectsuch as a μ opioid receptor agonist (e.g., morphine). Subjects that maybe treated by the compounds identified by the present invention includeboth human subjects and animal subjects (e.g., dogs, cats, horses,cattle) for veterinary purposes.

Thus, as noted above, further aspects of the present invention includeactive compounds produced or identified by the methods describedhereinabove and pharmaceutical formulations of the same (e.g., saidcompound in a sterile pyrogen-free saline solution), along with the useof such compounds for the preparation of a medicament for thepotentiation of the activity of μ opioid receptor agonists such asmorphine, and/or for the control of pain, in a subject in need thereof,either alone or in combination with a μ opioid receptor agonist such asmorphine.

In addition to morphine, other μ opioid receptor agonists, typicallyopiates, that may be used in conjunction with the present inventioninclude, but are not limited to, codeine, oxycodeine, hydromorphone,diamorphine, methadone, fentanyl, sufentanil, buprenorphine, meperidine(Demerol®), etc.

The active compounds described above may be combined with apharmaceutical carrier in accordance with known techniques to provide apharmaceutical formulation useful carrying out the methods describedabove. See, e.g., Remington, The Science And Practice of Pharmacy (9hEd, 1995). In the manufacture of a pharmaceutical formulation accordingto the invention, the active compound (including the physiologicallyacceptable salts thereof) is typically admixed with, inter alia, anacceptable carrier. The carrier must, of course, be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the patient. The carrier may be a solidor a liquid, or both, and is preferably formulated with the compound asa unit-dose formulation, for example, a tablet, which may contain from0.01 or 0.5% to 95% or 99% by weight of the active compound. One or moreactive compounds may be incorporated in the formulations of theinvention, which may be prepared by any of the well known techniques ofpharmacy consisting essentially of admixing the components, optionallyincluding one or more accessory ingredients.

The formulations of the invention include those suitable for oral,rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces), the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound of thepresent invention+, or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

Formulations suitable for topical application to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, transdermal enhancers, and combinationsof two or more thereof.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Formulationssuitable for transdermal administration may also be delivered byiontophoresis (see, for example, Pharmaceutical Research 3(6):318(1986)) and typically take the form of an optionally buffered aqueoussolution of the active compound. Suitable formulations comprise citrateor bis\tris buffer (pH 6) or ethanol/water and contain from 0.1 to 0.2 Mactive ingredient.

Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder.

The present invention is explained in greater detail in the followingnonlimiting Examples. The examples which follow are set forth toillustrate the present invention, and are not to be construed aslimiting thereof. As used herein, βarr2-GFP=βarrestin2 green fluorescentprotein; GFP=green fluorescent protein; GPCR=G protein-coupled receptor;βARK=beta adrenergic receptor kinase; GRK=G protein-coupled receptorkinase; β2AR=β2 adrenergic receptor; HEK-293=human embryonic kidneycells; DMEM=Dulbecco's modified Eagle medium; and MEM=Minimal EssentialMedium.

EXAMPLE 1 Materials and Methods

Materials: Isoproterenol was obtained from Sigma RBI. Anti-mouseantibody was obtained from Sigma Chemicals or Molecular Probes. Mousemonoclonal antibody against the 12CA5 epitope was obtained fromBoehringer Mannheim. Cell culture media was obtained from Mediatech andfetal bovine serum from Atlanta Biologicals. Physiological buffers werefrom Gibco-Life Technologies Inc. Restriction enzymes were obtained fromPromega or New England Biolabs, T4 ligase was from Promega, and Hot TubDNA polymerase from Amersham. Commercially available plasmids containingvariants of Green Fluorescent Protein were obtained from Clontech.

Cell Culture and Transfection: HEK-293 and COS cells were maintained andtransfected as described by Barak et al., Mol. Pharm. 51:177 (1997).Cells containing both β2 adrenergic receptor and βarrestin constructswere transfected with between 5-10 μg of receptor cDNA in pcDNA1/AMP and0.5-1 μg of βarr2-GFP cDNA per 100 mm dish. GRKs were expressed using 5μg of transfected cDNA in pcDNA1/AMP per dish.

Confocal Microscopy: HEK-293 cells transfected as described above wereplated onto 35 mm dishes containing a centered, 1 cm well formed from ahole in the plastic sealed by a glass coverslip. Primary and secondaryantibody labeling of live cells were performed at 37° C. for 30 minutesin media without serum in a 5% CO₂ incubator. Cells were washed threetimes between applications. Cells plated as above in MEM or DMEMbuffered with 20 mM Hepes were viewed on a Zeiss laser scanning confocalmicroscope.

Sequestration: Flow cytometry analysis was performed using techniquesknown in the art, as described in Barak et al., J. Biol. Chem. 269:2790(1994).

EXAMPLE 2 Construction of βarrestin2-GFP Plasmid

βarrestin2 cDNA in the plasmid pCMV5 was used as a template.Oligonucleotide primers surrounding a distal XhoI restriction site andthe C-terminal stop codon of βarrestin2 were used to replace the stopcodon with an in frame BamHI restriction site by directed mutagenesis(Valette et al. Nucleic Acids Res. 17:723 (1989); Attramadal et al., J.Biol. Chem. 267:17882 (1992); Lohse et al., Science 248:1547 (1990)).The XhoI, BamHI segment was isolated. This segment was ligated to theN-terminal portion of βarrestin cDNA (cut from pCMV5 by SacI and XhoI)in the polylinker of a plasmid that had been previously digested withSacI and BamHI and that contained S65T-Green Fluorescent Protein distaland in frame to the site of βarrestin cDNA insertion. Lohse et al.,Science 248:1547 (1990). The resulting βarrestin-GFP construct wasisolated following insertion and growth in E. coli. Constructs wereverified by sequencing.

A linear model of the βarrestin2/S65T-GFP conjugate is provided in FIG.1.

EXAMPLE 3 Characterization of βarr2-GFP Expressed by HEK-293 Cells

Homogenates of HEK-293 cells transformed with the plasmid of Example 2were studied using known Western Blot techniques. The results showedthat HEK-293 cells expressed both endogenous βarrestin and the βarr2-GFPconjugate.

Western Blots of homogenates of HEK-293 cells transfected with theplasmid of Example 2 and expressing βarr2-GFP were performed. An equalamount of homogenate material was loaded into each of two lanes (FIG.2A). The left lane was exposed to anti-βarrestin antibody (Menard etal., Mol. Pharm. 51:800 (1997)), whereas the right lane was exposed to amouse monoclonal antibody against GFP. The βarr2-GFP fusion protein isapproximately 50% larger than βarrestin2, and would thus be expected tomigrate more slowly than βarrestin on SDS-Page.

Exposure to anti-βarrestin antibody revealed multiple bars (left lane);exposure to anti-GFP monoclonal antibody revealed a single bar (rightlane). The position of endogenous cellular βarrestin2 is indicated bythe intermediate bar in the left lane (βarr2). The heavy band just below71,000 on the left lane (βarr2-GFP) is mirrored by a similar band in theright lane. In contrast, no band corresponding to endogenous cellularβarrestin 2 is observed with anti-GFP antibody exposure. The treatmentof the right lane with anti-GFP antibody demonstrated that the slowerband labeled by anti-βarrestin antibody contained GFP.

EXAMPLE 4 Biological Activity of βarrestin-GFP Conjugate

βarrestin activity can indirectly be assessed by measuring its effect onreceptor sequestration (see Menard et al., Mol. Pharm. 51:800 (1997);Ferguson et al., Science 271:363 (1996)). The β2AR normally sequesterspoorly in COS cells, and this has been correlated to the relatively poorexpression of endogenous βarrestins (see Menard et al. Mol. Pharmacol.51:800 (1997); Ferguson et al, Science 271:363 (1996)). Overexpressionof exogenous βarrestin enhances β2AR sequestration in these cells. Todemonstrate that the βarr2-GFP conjugate is a biologically activeβarrestin, COS cells overexpressing βarr2-GFP were examined foraugmentation of β2AR internalization, compared to the augmentation ofβAR2 seen with the overexpression of βarrestin2. Results are shown inFIG. 2B.

Using epitope tagged βAR2 receptors, sequestration of βAR2 was studiedin COS cells overexpressing either (1) exogenous βarrestin2 or (2) theβarr2-GFP conjugate. FIG. 2B shows the sequestration of β2AR in COScells with and without overexpressed βarrestin2 (left two bars) and withand without overexpressed βarr2-GFP (right two bars). Agonist mediatedβ2AR sequestration increased from 15.+−0.7% to 39.+−0.5% in the presenceof overexpressed βarrestin2; overexpression of βarr2-GFP similarlyincreased agonist mediated β2AR sequestration from 25.+−0.4% to58.+−0.1%. Wild type βarrestin2 and βarr2-GFP enhanced β2ARsequestration equally well above control levels, producing a 2.5 and 2.4fold increase in β2AR sequestration, respectively.

The above results indicated that the βarr2-GFP conjugate acts as abiologically active arrestin.

EXAMPLE 5 Agonist Mediated Translocation of βarr2-GFP

Agonist mediated translocation of the βarr2-GFP chimera from cellcytosol to membrane was studied using HEK-293 and COS cells transfectedwith plasmids containing cDNA for the β2AR receptor and for theβarr2-GFP conjugate.

HEK-293 and COS cells were transfected with plasmids containing 10 μg ofcDNA for β2AR and 0.5-1.0 μg for βarr2-GFP. Cells were assessed usingconfocal microscopy to detect the inherent intracellular fluorescence ofGFP.

Transfected HEK-293 cells are shown in FIG. 3A, where panel 1 depictscells prior to the addition of βAR2 agonist, and panel 2 depicts cellsfollowing the addition of agonist. Transfected COS cells are shown inFIG. 3B, where panel 1 depicts cells just prior to the addition of βAR2agonist, and panel 2 depicts cells ten minutes after the addition ofagonist.

As shown in FIG. 3A, βarr2-GFP distribution in HEK-293 cells wasinitially cytosolic (panel 1). No significant nuclear or membraneenhancement was apparent. Following the addition of the βAR2 agonistisoproterenol to the cell medium, the real-time agonist-mediatedredistribution of βarr2-GFP was viewed using confocal microscopy. Tenminutes after isoproterenol addition (saturating concentrations),enhancement of membrane fluorescence was seen with a concomitant loss ofcytosolic fluorescence, indicating that the βarr2-GFP distribution hadshifted to the membrane (panel 2). These results establish that inHEK-293 cells containing the β2AR, βarr2-GFP expressed by the cell istranslocated from cytosol to membrane following the addition of a βAR2agonist. Exposure of the test cells to GPCR agonist enhanced membranebound fluorescence ten-fold over that seen prior to agonist exposure.

As shown in FIG. 3B, βarr2-GFP distribution in COS cells was initiallycytosolic (panel 1). No significant nuclear or membrane enhancement wasapparent. Following the addition of the βAR2 agonist isoproterenol tothe cell medium, the real-time agonist-mediated redistribution ofβarr2-GFP was viewed using confocal microscopy. Ten minutes afterisoproterenol addition (saturating concentrations), enhancement ofmembrane fluorescence was seen with a concomitant loss of cytosolicfluorescence, indicating that the βarr2-GFP distribution had shifted tothe membrane (panel 2). These results establish that in COS cellscontaining the β2AR, βarr2-GFP expressed by the cell is translocatedfrom cytosol to membrane following the addition of a βAR2 agonist.

Comparing FIGS. 3A and 3B shows that the fluorescent signal is reducedin COS cells as compared to HEK cells, reflecting the lower efficiencyof sequestration of the β2AR in COS cells. However, even in COS cellsthe shift of βarr2-GFP in COS cells from cytosol to membrane followingthe addition of βAR2 agonist is clearly discernible due to thefluorescence of the GFP moiety.

The above experiments with COS and HEK-239 cells were reproduced exceptthat the βAR2 antagonist propranolol was added to the cell medium. Usingconfocal microscopy to visually track βarr2-GFP in the cell in realtime, as above, indicated that no shift in βarr2-GFP from cytosol tomembrane occurred in response to a βAR2 antagonist. As shown in FIG. 6E,addition of an agonist (middle panel) resulted in translocation ofβarr2-GFP from cytosol to membrane; subsequent addition of an antagonist(right panel) reversed the translocation (compare to control, leftpanel).

Biochemical evidence indicates that βarrestins are predominantlycytosolic proteins. Ferguson et al, Can. J. Physiol. Pharmacol. 74:1095(1996). The present results confirm that βarr2-GFP is distributedthroughout the cytosol and excluded from the nucleus. These data alsoestablish that βarr2-GFP is not predominantly compartmentalized at theplasma membrane in the absence of agonist, but that upon exposure to anagonist the cellular βarr2-GFP shifts to the membrane. The presentresults further indicate that the shift of the βarr2-GFP conjugate inresponse to the addition of a G protein coupled receptor agonist can bedetected optically as an enhancement of membrane fluorescence and/or aconcomitant loss of cytosolic fluorescence, and that this response israpidly observed.

EXAMPLE 6 Intracellular βarr2-GFP Targets Membrane Receptors

FIG. 4 shows the time course of βarr2-GFP redistribution to plasmamembrane 12CA5(HA) tagged β2AR in HEK-293 cells, as shown by confocalmicroscopy. The present example demonstrates that β2ARs are the targetof intracellular βarr2-GFP conjugate proteins. HEK-293 cells containing12CA5(HA) tagged β2AR receptors were studied. The receptors in theHEK-293 cells were reorganized into plasma membrane clusters (Row A) bycrosslinking with a mouse monoclonal antibody directed against anN-terminal epitope, followed by Texas Red conjugated goat anti-mouseantibody. In FIG. 4, the three panels of Row A show the same HEK-293cell with βAR2 receptors reorganized into plasma membrane clusters.

HEK-293 cells were then exposed to agonist (isoproterenol added to cellmedium, as above); the three panels of Row B in FIG. 4 were takenconsecutively after agonist addition (left to right, at 0, 3 and 10minutes post agonist addition). The real-time redistribution ofβarr2-GFP to the receptors over a ten minute time period is thusdemonstrated by comparing the panels of Row A and Row B of FIG. 4. InFIG. 4, arrows indicate areas of colocalization and the bar=10 microns.

FIG. 4 demonstrates that the geometry of the agonist-induced timedependent translocation of βarr2-GFP to the plasma membrane mimicked thedistribution of pre-aggregated β2ARs. This indicates that the primarysite targeted by βarrestin is the β2AR or a closely associatedcomponent.

EXAMPLE 7 Intracellular βarr2-GFP Targets Membrane Receptors

It has been postulated that phosphorylation of GPCRs by GRKs facilitatesdesensitization by increasing their affinity for βarrestins. Gurevich etal., J. Biol. Chem. 268:16879 (1993); Gurevich et al, J. Biol. Chem.268:11628-11638 (1993); Ferguson et al., Can. J. Physiol. Pharmacol.74:1095 (1996). When expressed in HEK-293 cells and exposed to agonist,mutant Y326A-β2ARs are not significantly phosphorylated by endogenousGRKs. Barak et al., Biochem. 34:15407 (1995); Ferguson et al., J. Biol.Chem. 270:24782 (1995). This phosphorylation impairment in Y326A-βAR2sis reversed by overexpression of GRKs in the same cell. Menard et al.,Biochem. 35:4155 (1996). The Y326A mutant receptor was used toinvestigate βarrestin affinity in vivo; the effect of overexpressed GRKon the Y326A-B2AR interaction with βarr2-GFP was shown.

Y326A-β2AR and βarr2-GFP were co-transfected into HEK-293 cells, in theabsence and presence of co-transfected GRK. If phosphorylation of GPCRsby GRKs facilitates desensitization by increasing their affinity forβarrestins, then overexpression of GRK would result in a noticeabledifference in βarr2-GFP translocation.

FIG. 5 shows the influence of overexpressed GRK on the redistribution ofβarr2-GFP in HEK-293 cells expressing the Y326A phosphorylation impairedβ2AR. Cells without (Row A) and with (Row B) overexpressed GRKs wereexposed to agonist, and the real-time redistribution of βarr2-GFP wasobserved. Without added GRK, βarr2-GFP translocation in response toagonist proceeded poorly, as shown in Row A of FIG. 5. βarr2-GFPtranslocation in cells containing overexpressed GRK (Row B) was morerobust, indicating an increased affinity of βarr2-GFP for receptor andthe relationship of phosphorylation and βarrestin activity.

EXAMPLE 8 Testing of Additional Receptors in the βAR/rhodopsin Subfamily

Twelve different members of the βAR/rhodopsin subfamily of GPCRs havebeen studied. Cells expressing a particular GPCR, and containingβarrestin-GFP chimeric proteins were exposed to known agonists for theGPCR being studied. In each case, an observable translocation of theβarrestin-GFP chimeric proteins from the cell cytosol to the cellmembrane was produced within minutes following addition of the GPCRagonist (data not shown).

EXAMPLE 9 Production of βArrestin Knockout Mice

Because GPCRs, such as the substance P receptor and the opioidreceptors, participate in processing the sensation of pain, wecharacterized analgesic responses through the μ opioid receptor (μOR) inmice lacking βarrestin-2. In the clinical setting, morphine is currentlythe most effective drug for alleviating intense and chronic pain. Theantinociceptive (blocking of pain perception) actions of morphine aremediated through stimulation of the μOR, as demonstrated by the lack ofmorphine analgesia observed in knock out mice deficient in the μOR(H.Matthes et al., Nature 383, 819 (1996). B. Kieffer, Trends Pharmacol Sci20, 19 (1999); 1. Sora et al., Proc Nad Acad Sci USA 94, 1544 (1997)).Nevertheless, the neuronal signaling mechanisms mediating analgesiathrough μORs and morphine remain poorly understood. Moreover, thecontribution of GPCR desensitization to the onset and duration ofanalgesia has been unclear.

βarrestin-2 knockout (βarr2-KO) mice were generated by inactivation ofthe gene by homologous recombination. A bacteriophage A library of mouse129SvJ genomic DNA (Stratagene, La Jolla, Calif.) was screened with therat βarr2 cDNA (H. Attramadal et al., J. Biol. Chem. 267,17882 (1992)).Positive phages were identified and analyzed by restriction digest. A12-kb βarr2 fragment was digested with Bam HI, subcloned intopBluescript KS(−) and sequenced. The targeting vector was assembled byblunt-end ligation of a pHSV-TK cassette (from pIC19R/MCI-TK, M. R.Capecchi, University of Utah), a 2.8-kb Nco I-Bam HI βarr2 fragment, apGK-neo cassette (from plasmid pD383, R. Hen, Columbia University) whichreplaced the 0.8 kb Bam HI-Hind III fragment of βarr2, and a 4.5 kb HindIII βarr2 fragment into pBluescript KS(−). This targeting vector waslinearized with Not I and was electroporated into mouse embryonic stemcells. Genomic DNA from transfectants resistant to G418 and gancyclovirwere isolated and screened by Southern (DNA) blot analysis using a 0.2kb 5′ external βarr2 probe and a 0.3 kb 3′ external βarr2 probe.Chimeric animals were generated by microinjecting these ES cells intoC57BL/6 blastocysts. Five chimeric male pups were obtained and matedwith C57BL/6 females. Germline transmission was confirmed by Southernblotting. Heterozygous, offspring were intercrossed to obtain homozygousmice. Wild-type and mutant mice used in this study were age-matched, 3to 5 month old, male siblings. For protein immunoblot analysis, wholecell lysates were prepared by polytron homogenization in lysing buffer[10 mM Tris (pH 7.4), 5 mM EDTA, 1 protease inhibitor tablet/10 mL(Roche Molecular Biochemicals, Indianapolis, Ind., USA), 1% nonidet-40].Polyacrylamide gels were loaded with 25 μg protein/lane and equivalentprotein loading was confirmed by Ponceau S staining of the gels. Aftertransfer to polyvinyldifluoride (PVDF) membranes, proteins were blottedwith polyclonal antibodies to βarrestin-2 or βarrestin-1 [H. Attramadalet al., J. Biol. Chem. 267, 17882 (1992)]. Bands were visualized withsecondary antibody conjugated to horseradish peroxidase and an enhancedchemiluminescence detection system (Amersham, Piscataway, N.J.). Allexperiments were conducted in accordance with the NIH guidelines for thecare and use of animals.

EXAMPLE 10 Identification of βArrestin Knockout Mice

Mice lacking βarrestin-2 were identified by Southern DNA blot analysis(FIG. 7A) and the absence of βarrestin-2 was confirmed by proteinimmunoblotting of extracts from brainstem, periaqueductal gray (PAG)tissue, spleen, lung and skin (FIG. 7B). Wild-type, heterozygous, andhomozygous mutant mice had similar amounts of βarrestin-1 in the brainregions examined (FIG. 7B), arguing against compensatory upregulation ofβarrestin-1 in the absence of βarrestin-2. The βarr2-KO mice were viableand had no gross phenotypic abnormalities. However, after administrationof morphine, obvious differences became apparent between the genotypes.

EXAMPLE 11 Evaluation of Morphine-Induced Antinociception in βarrestinKnockout Mice

Morphine-induced antinociception was evaluated by measuring responselatencies in the hot plate test. We used a dose of morphine (10 mg/kg)and route of administration (s.c.) well established to induce analgesiain many strains of mice (F. Porreca et al., J Pharmacol Exp Ther 230,341 (1984). J. Belknap et al., Physiol Behav 46, 69 (1989). A Gardmarket al., Pharmacol Toxicol 83, 252 (1998). G. Elmer et al., Pain 75, 129(1998)). The analgesic effect of morphine was significantly potentiatedand prolonged in the knockout mice as compared to that in theirwild-type littermates (FIG. 8). Such robust responses to morphine werenot only absent in the wild-type liftermates (FIG. 8) but also in theparental mouse strains (C57BL/6 and 129SvJ) used to generate thisknockout. Four hours after the morphine injection, βarr2-KO mice stillexhibited significant analgesia (% maximum possible effect=31±0.4%);whereas, in control wild-type littermates, the analgesic effects of thesame dose of morphine waned after about 90 minutes. βarr2+/−mice werenearly as responsive to morphine as the βarr2-KO mice; however, this mayreflect the imposed limit of the hot plate assay (30 seconds), which isdesigned to prevent prolonged exposure of the mice to pain. Basalresponses to the hot plate did not differ between genotypes (wild type:6.2±0.3 sec., n=25; βarr2-KO: 6.1±0.4 sec., n=27). The differences inmorphine-induced analgesia between the genotypes are unlikely to be dueto pharmacokinetic differences in morphine metabolism, because theconcentrations of morphine in blood, as determined by mass spectroscopyanalysis, did not differ between wild type and βarr2-KO mice 2 hoursafter morphine injection (Mice were injected with morphine (10 mg/kg,subcutaneous). After 30 minutes or 2 hours, wild-type mice were killedand blood was collected in vials containing sodium fluoride andpotassium-oxalate. Morphine concentration in blood samples pooled from 3mice per sample were 1,500 ng/mL after 30 min., and 83 ng/mL blood after2 hours as measured by mass spectroscopy analysis [Occupational TestingDivision of LabCorp, Inc., Research Triangle Park, North Carolina, USA].In similar experiments, βarr2-KO mice had a concentration of 93 ng/mL inthe blood after 2 hours.

EXAMPLE 12 Evaluation of Low Dosage Morphine in βarrestin Knockout Mice

Lower doses of morphine were also tested in these assays. Even at dosesof morphine (1 mg/kg, s.c.) that were sub-analgesic in wild type mice,βarr2-KO animals displayed a significant increase in their nociceptivethresholds (FIG. 9). At 30 minute intervals, immediately following theantinociception test, mice were given repeated cumulative doses ofmorphine resulting in final concentrations of 5, and 10 mg/kg (I. Soraet al., Proc Natl Acad Sci USA 94, 1544 (1997)). At the highestcumulative dose, mice reached similar levels of antinociception as seenin FIG. 8, in which this amount of morphine was administered in a singleinjection. At every dose, the βarr2 KO animals experienced greaterantinociception after morphine treatment than did their wild-typelittermates.

EXAMPLE 13 Evaluation of Morphine Antagonists in βarrestin Knockout Mice

To test whether the analgesic effects of morphine were mediated byactions at the μOR, mice were treated with various antagonists. Naloxone(2.5 mg/kg, subcutaneous injection) which immediately reverses theeffects of opiates, was given 30 minutes after morphine (10 mg/kg).Naltrindole [P. Portoghese et al., J Med Chem. 88, 1547 (1990)] wasgiven 20 minutes before morphine, and norbinaltorphimine (A. Takemori etal., J Pharmacol Exp Ther 246, 255 (1988)) was given 1 hour beforemorphine (H. Matthes et al., J Neurosci 18, 7285 (1998)). Naloxone, awell-established OR antagonist, was administered to the same mice,immediately after measuring the antinociceptive effects of morphine (10mg/kg). Naloxone (2.5 mg/kg, s.c.) completely reversed the effects ofmorphine in both the wild-type and βarr2-KO animals within 10 minutes.However, the σ and κ OR-selective antagonists naltrindole (2.5 mg/kg,s.c.) and nor-binaltorphimine (5 mg/kg s.c.) did not inhibit analgesiain wild type nor βarr2-KO mice (data not shown). The morphine dosedependency of the antinociceptive response and the reversal of theeffects with naloxone suggest that the potentiated and prolonged effectsin mice that lack βarrestin-2 result from stimulation of the μOR.

EXAMPLE 14 Body Temperature Measurements in Wild-Type and βarrestinKnockout Mice

Wild-type and βarr2-KO mice were also evaluated for changes in bodytemperature (M. Adler et al., Annu Rev Pharmacol Toxicol 28, 429 (1988).Rectal body temperatures were determined with a digital thermometer [F.Fumagalli et al., J Neurosci 18, 4861 (1998)] (TH8, Physitemp, Clifton,N.J., USA). The probe was inserted into the rectum and maintained untilthe temperature reading stabilized. No significant differences in basalbody temperature were found between genotypes, however βarr2-KO miceexperienced a greater drop in body temperature after morphine treatmentthan did wild-type (FIG. 10). This greater decrease in temperature alsopersisted longer than that in their wild type littermate controls.

EXAMPLE 15 Radioligand Binding Assays

To investigate whether the μOR population was altered in the KO mice,radioligand binding analysis on membranes prepared from different brainregions was performed. Brain regions were dissected and immediatelyfrozen in liquid nitrogen and were stored at −80° C. for less than 1week before use. Samples were placed on ice and homogenized by polytronin membrane preparation buffer [50 mM Tris (pH 7.4), 1 mM EDTA, 3 mMMgCl₂] and crude membranes were prepared by centrifugation at 20,000×gfor 15 min at 4° C. Membranes were resuspended in either 50 mM Tris-HCl(pH 7.4) for radioligand binding assays or in assay buffer [50 mMTris-HCl (pH 7.4), 100 mM NaCl, 3 mM MgCl₂, 0.2 mM EDTA] containing 10μM GDP for [³⁵S] GTPγS binding assays. For both binding assays,reactions were terminated by rapid filtration over GF/B filters(Brandel, Inc., Gaithersburg, Md.) using a Brandel cell harvester(Brandel, Inc., Gaithersburg, Md.). Filters were washed 3 times with icecold 10 mM Tris-HCl (pH 7.4) and then counted in a liquid scintillationcounter. Hypothalamus, brain stem, and periaqueductal gray (PAG) regionswere chosen because they contain μORs and are implicated in theregulation of pain and body temperature (D. Mayer and D. Price, Pain 2,379 (1976). T. Yaksh et al., Prog Brain Res 77, 371 (1988). D. J.Smith., et al., Eur J Pharmacol 156, 47 (1988)). Data are given in TableI. Saturation binding studies with ³H-naloxone, at concentrations thatpreferentially label the μOR, revealed a single high affinity bindingsite, which represents the μOR. The number and affinity of μORs did notsignificantly differ between the two genotypes in any of the brainregions examined.

TABLE I H-Naloxone binding in brain regions of Wild Type and Knockoutmice.¹ Wild Type βarr2-Knockout B_(MAX) K_(D) B_(MAX) K_(D) Brain region(fmol/mg) (nM) (fmol/mg) (nM) PAG 132 ± 9  4.0 ± 0.1 144 ± 13 3.0 ± 0.8Brainstem 49 ± 7 1.5 ± 0.2 54 ± 9 3.0 ± 0.8 Hpothalamus 103 ± 18 6.2 ±1.6 89 ± 8 3.8 ± 0.2 ¹Saturation binding assays were performed onmembranes from different brain regions (50-100 μδ/tube) with increasingconcentrations of ³H-naloxone (0-12 nM, 52.5 Ci/mmol, Amersham,Piscataway, New Jersey, USA). Nonspecific binding was determined in thepresence of 10 μM naloxone. Membranes were incubated at 25° C. for Ihour. Binding parameters were determined via Scatchard analysis ofspecific binding. Data are the mean ± S.E.M. of 3-4 experimentsperformed in duplicate.

Additional evidence for increased sensitivity of the μOR in βarr2-KOanimals was obtained in biochemical experiments. We measuredagonist-stimulated binding of [³⁵S]GTPγS to G proteins in isolatedmembranes the most proximal manifestation of GPCR activation (D. Selleyet al., Mol Pharmacol 51, 87 (1997)). Because morphine acts in vitro tostimulate μ, σ, and κ opioid receptors, the μOR-selective agonist,[D-Ala², MePhe⁴, Gly⁵-ol]enkephalin (DAMGO), was used to specificallyactivate G protein coupling to μORs. DAMGO stimulated more [³⁵S]GTPγSbinding in membranes derived from βarr2-KO mice than in those derivedfrom wild-type littermates (FIG. 11). Similar results were also obtainedin brainstem membranes (data not shown). The amount of Gα proteins(G_(i/o/z)) as determined by protein immunoblotting, did not varybetween the genotypes (data not shown). These observations suggest thatthere is enhanced coupling of μORs to G proteins in tissues derived fromβarr2-KO mice. Although the enhanced analgesia induced by morphine mayinvolve complex neurological-signaling, this biochemical evidencesupports the interpretation that the enhanced physiologicalresponsiveness in the knockout animals results from increasedsensitivity of signaling by the μOR.

These studies demonstrate in an animal model that the absence ofβarrestin-2 can affect the efficacy of GPCR activation. In transfectedcultured cells, the degree of β₂-adrenergic receptor signaling isdependent upon the cellular complement of GRK2 and GRK3 and βarrestins(L. Menard et al., Mol Pharmacol 51, 800 (1997); S. Mundell et al.,Biochemistry 38, 8723 (1999)). These observations, along with thosepresented here, directly support the proposed role of βarrestin-2 inpreventing further receptor-G protein coupling and mediatingdesensitization of the GPCR. Moreover, βarrestins are not only involvedin the dampening of GPCR responsiveness after agonist stimulation, butalso influence the sensitivity of the response.

The simplest interpretation of these results is that μOR signaling isregulated by βarrestin-2. However, in transfected cells, morphine failsto induce the internalization of the μOR and a GFP-tagged βarrestin-2fails to translocate to μOR overexpressed in cell culture upon exposureto morphine (J. Arden et al., J Neurochem 65, 1636 (1995). D. Keith etal., J Biol Chem 271, 19021 (1996); J. Whistler and M. von Zastrow, ProcNatl Acad Sci USA 95, 9914 (1998); J. Zhang et al., Proc Natl Acad SciUSA 95, 7157 (1998)). Interestingly, these in vitro studies have beenconducted with the rat μOR or the mouse MOR1 which are not particularlyrich in potential phosphorylation sites. Several splice variants of theμOR are present in mouse brain that contain several potentialphosphorylation sites (Y. Pan et al., Mol Pharmacol 56, 396 (1999)).Some of these isoforms can contribute to morphineinduced analgesia. Theinvolvement of these receptors might explain the differences between thein vitro studies and those with the βarr2-KO mice.

The βarr2-KO mice were very similar in phenotype to their wild typelittermates and other GPCR-directed drugs did not necessarily elicitdifferent responses between the genotypes. For example, locomotorresponses to dopamine receptor stimulation by cocaine and apornorphinewere not enhanced (data not shown). These observations suggest thatvarious GPCRs are differentially affected by the loss of βarrestin-2.Other regulatory elements, such as GRKs or βarrestin-1, could compensatefor the lack of βarrestin-2, or the receptors could vary in theirrequirement for βarrestin interaction for their regulation.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A conjugate comprising a β-arrestin protein and a label, wherein saidlabel is capable of indicating intracellular translocation and/ordistribution of said β-arrestin protein, wherein said β-arrestin iscapable of interacting with a G protein coupled receptor (GPCR).
 2. Theconjugate according to claim 1, wherein said conjugate is a fusionprotein.
 3. The conjugate of claim 1, wherein said label is an opticallydetectable label.
 4. A conjugate comprising a β-arrestin-2 protein and alabel, wherein said label is capable of indicating intracellulartranslocation and/or distribution of said β-arrestin-2 protein, whereinsaid β-arrestin-2 is capable of interacting with a G protein coupledreceptor (GPCR).
 5. The conjugate according to claim 1, wherein saidlabel is a fluorescent label.
 6. The conjugate according to claim 1,wherein said label is a colorimetric label.
 7. The conjugate accordingto claim 1, wherein said label is a radioactive label.
 8. The conjugateaccording to claim 1, wherein said label is an electron-dense label. 9.The conjugate according to claim 5, wherein said fluorescent label isGreen Fluorescent Protein (GFP).